Plasmids for gene editing

Abstract

The present invention pertains to single plasmid systems comprising sequences encoding programmable proteins, one or more guides, optionally donor polynucleotides, and optionally anti-CRISPR molecules, for gene editing. These plasmid systems allow for genomic engineering of bacterial strains that are difficult to transform and increase the efficiency of genomic engineering in tractable strains. Additionally, the single plasmids can be configured to provide for the transformation of a number of different bacterial strains using the same plasmid.

1 . A plasmid comprising:
 a sequence encoding a programmable CRISPR-associated (Cas) protein operably linked to an inducible promoter;   a guide polynucleotide capable of forming a complex with the Cas protein upon expression of the Cas protein, wherein the complex is capable of targeting a selected target site;   a first polynucleotide sequence homologous to a 3′ region adjacent to the selected target site;   a second polynucleotide sequence homologous to a 5′ region adjacent to the selected target site;   a sequence for a selectable marker; and   control elements that provide for expression of the plasmid sequences in a selected host cell.   
     
     
         2 . The plasmid of  claim 1 , wherein the first polynucleotide sequence and second polynucleotide sequence are operably linked 5′ and 3′, respectively, to a donor polynucleotide. 
     
     
         3 . The plasmid of  claim 1 , wherein the Cas protein comprises a catalytically active Cas endonuclease capable of producing a double-strand break at the selected target site. 
     
     
         4 . The plasmid of  claim 3 , wherein the Cas endonuclease comprises a Cas9. 
     
     
         5 . The plasmid of  claim 1 , wherein the programmable Cas protein comprises a nickase capable of producing a single-strand break at the selected target site. 
     
     
         6 . The plasmid of  claim 5 , wherein the nickase comprises a Cas9 nickase (nCas9). 
     
     
         7 . The plasmid of  claim 1 , wherein the programmable Cas protein comprises a catalytically inactive Cas protein (dCas) capable of binding to the selected target site but incapable of producing a double-strand or single-strand break at the selected target site. 
     
     
         8 . The plasmid of  claim 7 , wherein the dCas comprises dCas9. 
     
     
         9 . The plasmid of  claim 1  further comprising a sequence encoding an anti-CRISPR molecule operably linked to a promoter, wherein the anti-CRISPR molecule is capable of inhibiting the function of the programmable Cas protein. 
     
     
         10 . The plasmid of  claim 9 , wherein the anti-CRISPR molecule is selected from the group consisting of an AcrIIA1, an AcrIIA1-2, an AcrILAZ, an AcrIIA4, and an AcrIIA5. 
     
     
         11 . The plasmid of  claim 9 , wherein a constitutive promoter is operably linked to the sequence encoding the anti-CRISPR molecule. 
     
     
         12 . The plasmid of  claim 1 , wherein the inducible promoter operably linked to the sequence encoding the programmable Cas protein comprises an inducible tetracycline promoter. 
     
     
         13 . The plasmid of  claim 1 , wherein the sequence for the selectable marker is capable of imparting antibiotic resistance to the host cell transformed with the plasmid. 
     
     
         14 .- 20 . (canceled) 
     
     
         21 . The plasmid of  claim 1 , wherein the control elements comprise two or more origins of replication. 
     
     
         22 . A prokaryotic host cell transformed with the plasmid of  claim 1 . 
     
     
         23 . The prokaryotic host cell of  claim 22 , wherein the prokaryotic cell comprises a Proteobacteria cell. 
     
     
         24 . The prokaryotic host cell of  claim 23 , wherein the prokaryotic cell comprises an  Escherichia coli  cell. 
     
     
         25 . The prokaryotic host cell of  claim 22 , wherein the prokaryotic cell comprises a Bacteroidetes cell. 
     
     
         26 . The prokaryotic host cell of  claim 25 , wherein the prokaryotic cell comprises a  Bacteroides  spp. 
     
     
         27 - 30 . (canceled) 
     
     
         31 . A method for editing a prokaryotic genome comprising:
 transforming a selected prokaryotic cell with the plasmid of  claim 1 ; and   culturing the cell under conditions whereby the components of the plasmid are expressed such that homologous recombination at the selected target site occurs,   thereby editing the prokaryotic genome.   
     
     
         32 .- 39 . (canceled) 
     
     
         40 . The method of  claim 31 , wherein the prokaryotic cell is transformed by a method selected from the group consisting of electroporation, chemical transformation, and conjugation.